Quorum sensing (QS) allows bacteria to behave as a group at high cell densities and is closely connected to virulence in many common pathogens. [1] This intercellular communication system is mediated by small molecule or peptide signals that diffuse out of or are secreted by bacteria into the local environment. As the population grows, the concentration of QS signal increases until a threshold is reached, whereupon the signal can productively bind to its cognate receptor. The activated receptor then modifies gene expression levels to let the bacteria adopt a community lifestyle, while simultaneously increasing production of the QS circuitry and thus amplifying the QS response. [1a]
For many pathogens, QS allows bacteria to amass a sufficiently high population prior to initiating virulence phenotypes, thereby increasing their chances to successfully infect a host. [1a, 2] The close connection been QS and bacterial infection, and perhaps more fundamentally, the chemical nature of QS signaling, has inspired considerable recent research to design chemical strategies to intercept QS pathways. [3] Many of these efforts have focused on the development of synthetic mimics of QS signals that can inhibit QS receptor:signal binding, for use as chemical probes to block virulence phenotypes, and to delineate basic QS mechanisms. [1a, 4, 5] Efforts include those directed to the common pathogen Staphylococcus aureus [4f-l, 4k, 4l, 6] and related species. [4j]
In the staphylococci, QS is controlled by the accessory gene regulator (agr) system. [5t, 7] In the case of S. aureus, this QS system controls the expression of over 100 virulence factors. [7b, 8] The S. aureus agr machinery is composed of four proteins, AgrA-D, and a signaling molecule (derived from AgrD) termed an autoinducing peptide (or AIP; FIG. 1A). [9]
AgrD is the precursor to the QS signal, which is processed by AgrB and secreted as the mature AIP (FIG. 1A). [5t, 7, 9a, 10] The S. aureus AIP is a small macrocyclic peptide (7-9 residues), containing a short N-terminal tail and a thiolactone bridge between an internal Cys side chain and the C-terminus. [5j, 9a] To date, four specificity subgroups of S. aureus have been characterized (groups I-IV), each defined by the unique peptide sequence of their AIPs (shown in FIG. 1B) and their target transmembrane receptor and histidine kinase, AgrC. [9a, 11] When a threshold extracellular AIP concentration is reached, the peptide signal binds and activates AgrC. AgrC then phosphorylates and thereby activates its partner response regulator, the transcription factor AgrA. [5o] AgrA subsequently targets several promoters, including P2 and P3. P2 induces transcription of the agr operon, and provides positive feedback for the autoinduction circuit. [9b] In turn, P3 drives transcription of RNA-III, which is a major regulator of virulence factor production in S. aureus. [12]
Blocking AgrC:AIP binding represents one strategy to attenuate QS signaling in S. aureus, and this approach has been the focus of considerable research. [4g, 4l, 5t, 7a, 7b] The initial discovery that AIP-I and AIP-II inhibit AgrC activity in non-self Agr specificity groups was followed by study of AIP derivatives with tail-truncations, sequential Ala scans, and other amino acid substitutions to identify key structure-activity relationships (SARs) dictating ligand and receptor activity. [5j-l, 5q, 7b] These early SAR studies suggested that the AIP ligand-binding site on AgrC was a hydrophobic cleft that could be a viable target for competitive inhibition with hydrophobic, peptidic ligands. [5l, 5m, 13] Additional screening with tail-truncated AIPs resulted in further refinement of the SARs for AgrC activation and inhibition, suggesting that the presence of only two hydrophobic C-terminal groups within ligands was required for AgrC binding, while additional contacts on the N-terminal tail were needed for receptor activation.[5l, 5n]
Tail-truncated AIP-II (t-AIP-II) was found to be quite potent amongst this class.[5l, 5n] Recently, similar SAR studies were extended to S. aureus AIP-III, which found a similar activity trend; namely, hydrophobic endocyclic residues were required for AgrC binding, while exocyclic tail contacts along with the hydrophobic motifs were required for activation. [4g-i, 4k, 4l] Studies with AIP-III and mimetics thereof identified a number of highly potent, pan-group AgrC inhibitors, with AIP-III D4A (IN(CAFLL) SEQ ID NO:6) being one of the most potent AgrC inhibitors reported to date. [4g]
Despite their potency, however, peptidic AgrC modulators possess several qualities that limit their utility as chemical tools. First, the AIP thiolactone bridges are hydrolytically unstable.[4l, 6a, 14] Second, while their macrocyclic framework renders AIPs more proteolytically stable than linear peptides, they are still susceptible to proteolysis.[4l, 15] Third, AIP-type peptides have relatively low water solubilities due to their hydrophobic structures. Fourth, these ligands are typically prepared using solid-phase synthesis techniques that do not lend themselves easily to large batch synthesis. The development of lactam-bridged AIP-III mimetics has begun to address some of these limitations; [4l] however, significant challenges certainly remain.
Thus, there is a need in the art for non-peptide, small molecule mimetics of AIPs that display enhanced stabilities and aqueous solubilities, reduced immunogenicity, and are amendable to larger scale synthesis relative to peptides.